Tuberculosis and the Tubercle Bacillus, Second Edition

Tuberculosis (TB) remains one of the leading causes of death by an infectious agent, accounting for approximately 1.3 million deaths per year ( 1 ). Despite its clinical significance, there are still significant gaps in our understanding of Mycobacterium tuberculosis pathogenesis and the host mechanisms that limit active disease to approximately 10% of those infected. Nevertheless, we continue to gain insight into the dynamic interplay between pathogen and host, with much of the focus centered on the lung microenvironment because this is the initial and primary site of infection. The lung as the initial “battlefield” provides unique challenges to both the host and pathogen because the host must balance the inflammatory response to limit the damage to lung tissue while inducing a sufficient immune response to control the infection. In contrast, the M. tuberculosis organism must avoid or circumvent the initial defensive barriers present within the respiratory tract to gain access to its host cell, the alveolar macrophage (AM). The AM response to infection as well as the reaction of other lung immune and nonimmune cells and noncellular components is critical to determining whether the host will directly eliminate the pathogen or will in concert with the acquired immune system develop a protective granulomatous response. In addition, since bacteria disseminate during the early events in infection, engagement of innate immune components outside of the lung is also critical in shaping the host response. These early host processes which constitute the innate immune system will be the focus of this article.

Cytokines are soluble, small proteins that are produced by cells and act in a largely paracrine manner to influence the activity of other cells. Currently, the term “cytokine” describes proteins such as the tumor necrosis factor family, the interleukins, and the chemokines. Virtually every nucleated cell can produce and respond to cytokines, placing these molecules at the center of most of the body’s homeostatic mechanisms ( 1 ). Much of our knowledge of the function of cytokines has been derived from studies wherein homeostasis has been disrupted by infection and the absence of specific cytokines results in a failure to control the disease process. In this context, infection with Mycobacterium tuberculosis has proven to be very informative and has highlighted the role of cytokines in controlling infection without promoting uncontrolled and damaging inflammatory responses ( 2 – 4 ). Herein, we focus on the key cytokine and chemokines that have been studied in the context of human TB using experimental medicine as well as M. tuberculosis infection of various animal models, including non-human primates (NHPs), mice, and rabbits. Perhaps the most important message of this review is that in a complex disease such as TB the role of any one cytokine cannot be designated either “good” or “bad” but rather that cytokines can elicit both protective and pathologic consequences depending on context.

Homeostasis in the immune system has the essential function to minimize deleterious immune-mediated inflammation caused by commensal microorganisms, immune responses against self and environmental antigens, as well as metabolic inflammatory conditions. Homeostatic regulation is enabled by regulatory T (Treg) cells that mediate immune suppression as a vital mechanism of negative regulation. Treg cells have the capacity to prevent not only potentially damaging autoimmune responses, but also protective immunity, and thus the number of Treg cells is a crucial determinant of the regulatory burden on the immune system. The presence of low numbers of Treg cells can trigger fatal autoimmunity, whereas having high numbers can cause overt immunosuppression. Specifically, this means that the combination of the overall number and specific subsets of regulatory cells maintains the order in the immune system by a process of imposing negative regulation on other cells in the immune system. In this review, we will discuss the role of the distinct regulatory immune cell subsets in the development of tuberculosis (TB).

Mycobacterium tuberculosis is one of the most successful pathogens with approximately 30% of the world’s population harboring the bacterium. Although some highly exposed individuals appear resistant to infection with M. tuberculosis ( 1 ), once an individual is infected, there is little evidence that the ensuing immune response leads to sterilizing immunity ( 2 ). Instead, the majority of individuals infected with M. tuberculosis (>90%) develop an asymptomatic chronic tuberculosis (TB) infection known as latent TB. During latent infection, activated host immune cells result in arrest of mycobacterial growth and control of disease progression. Active disease can develop from latent infection if the immune response is sufficiently suppressed, and, over their lifetime, this will occur in 5 to 10% of the latently infected individuals ( 3 ).

Progress toward developing new strategies to control the spread of Mycobacterium tuberculosis is limited by a poor understanding of the basic pathogenesis of post-primary tuberculosis (TB). Progress is being made in developing more rapid diagnostic assays and implementing new anti-TB drug combinations, but we are failing to answer key scientific questions that will further advance the development of new treatment and prevention strategies. In a recent statement, Dr. Anthony Fauci, head of the National Institute of Allergy and Infectious Diseases, said, “We need to better understand the delicate balance between the host and pathogen in the context of the entire biological system and this requires a radical and transformational approach.” M. tuberculosis has coevolved with its human host for centuries. The more recent emergence of antimicrobial drug-resistant strains represents an additional challenge to controlling the global spread of TB. No new TB vaccines have been shown to be more effective than the original bacillus Calmette-Guérin (BCG) developed over 100 years ago. The development of new anti-TB drugs lags far behind the need, and prospects for host-directed therapies have validated neither targets nor biomarkers.

Animal models are an integral part of the scientific process, reflecting the physiological and anatomical similarities between many animal species and human beings. In the context of infectious diseases, multiple animal models have been used to extend our understanding of their pathophysiology and the host response to them. This is the backbone also of vaccine research, producing vaccines against once-dreaded multiple diseases that in previous centuries claimed the lives of many millions of people. Animal models are also invaluable in designing therapies, particularly drugs, with which to combat these diseases.

Small-animal models have provided a vast amount of useful information about the nature of tuberculosis infection and its pathogenesis, the host response to it, and the activity of potential new vaccines and drugs. While it is possible that central mechanisms would have been eventually discovered directly in humans, there is no doubt that much of what we know about the immune response to the disease was substantially accelerated by studies in the mouse model, which took direct advantage of the literal explosion in the availability of reagents developed by the mainstream immunology field over the past 3 or so decades. These advances have allowed us to define in great detail the nature of the T-cell response to Mycobacterium tuberculosis and its various elements, including control of activation and cellular recruitment by cytokines and chemokines, as well as more practical applications in the form of using this inexpensive model to test vaccines and drugs.

Among the animal models of tuberculosis (TB), the non-human primates (NHPs), particularly rhesus macaques (Macaca fascicularis) and cynomolgus macaques (Macaca mulatta), share the greatest anatomical and physiological similarities with humans. Macaques are highly susceptible to Mycobacterium tuberculosis infection and manifest the complete spectrum of clinical and pathological manifestations of TB as seen in humans. Therefore, the macaque models have been used extensively for investigating the pathogenesis of M. tuberculosis infection and for preclinical testing of drugs and vaccines against TB. This review focuses on published major studies that exemplify how the rhesus and cynomolgus macaques have enhanced and may continue to advance global efforts in TB research.

Tuberculosis (TB) affecting domestic livestock, including cattle, goats, and deer, is predominantly caused by Mycobacterium bovis. The disease in cattle, defined as bovine TB, is a major economic animal health problem worldwide, costing U.S. $3 billion annually, with >50 million cattle infected ( 1 ). Costs from this disease are related to a reduction in productivity in severely affected animals, testing, culling of affected animals, movement controls, and restriction on trade. Goats and farmed deer are also readily affected by TB, causing economic losses due to trade limitations, culling of affected animals, and depopulation of herds ( 2 , 3 ). In addition, M. bovis affects feral deer, where it may establish a wildlife reservoir, for example, in white-tailed deer in the United States, often resulting in a source of infection to contiguous cattle herds ( 4 ). M. bovis has a wide host range affecting both domestic and wild animals, but infection of other livestock such as domestic pigs and sheep is relatively rare. M. bovis is also infectious to humans, and prior to mandatory pasteurization in many countries, M. bovis accounted for about one fourth of TB cases in children ( 5 ). Transmission of M. bovis to humans has been markedly reduced with the pasteurization of milk and the implementation of bovine TB control programs coupled with abattoir surveillance, although risks remain with the consumption of unpasteurized milk and cohabitation of infected animals with humans.

The need for a new tuberculosis (TB) vaccination strategy is clear when we consider the massive burden of TB disease in countries where universal infant bacille Calmette-Guérin (BCG) vaccination is practiced as part of the World Health Organization (WHO) Expanded Program on Immunization (EPI) ( 1 ). The lifetime risk for development of TB disease in these high-transmission settings is driven by multiple ongoing Mycobacterium tuberculosis exposure-re-exposure episodes, beginning in early childhood, leading to M. tuberculosis infection at an early age, and continuing throughout adulthood ( Fig. 1 ). Infants and very young children not only have a higher risk of progression from infection to disease, but have a higher risk of severe disease, including miliary TB and tuberculous meningitis ( 2 ). Since BCG vaccine is thought to offer protective efficacy of about 74% against all forms of TB disease in children, BCG vaccination is firmly entrenched in the EPI ( 3 ). However, although BCG vaccination is thought to offer modest protection against M. tuberculosis infection as defined by interferon-gamma (IFN-γ) release assay (IGRA) conversion, the majority of adults in high-TB-burden countries are M. tuberculosis-infected ( 4 ). In very high-transmission settings such as South Africa, more than three quarters of adolescents are M. tuberculosis-infected by the time they leave high school ( 5 , 6 ). Given that the rate of infection in some African countries may exceed 10% per annum ( 7 ), it is not surprising that while Africa is responsible for only 28% of the world’s new TB cases, 7 of the top 10 countries for TB incidence by population are in Africa, where HIV coinfection, a younger population demographic, and social disadvantage and disruption add susceptibility to TB disease ( 8 ). It is against this backdrop that we review the potential indications for TB vaccines, in search of a vaccination strategy that is safe and effective against all forms of TB disease in infants, children, and adults, including M. tuberculosis-infected and HIV-infected people.

Immunity to Mycobacterium tuberculosis is an interplay between the innate and adaptive immune response, both cellular and humoral. This interplay is not static but changes over time as we grow, age, and respond to our environment. Animal models enable examination of individual components of the immune response at distinct time points during the course of infection. This has enabled identification and understanding of key immune mechanisms for M. tuberculosis control. However, rational development of interventions, such as more effective vaccines and other host-directed therapies, has to take into consideration the enormous heterogeneity of the interactions between M. tuberculosis with human innate and adaptive immune responses, which are profoundly influenced by genetic variation, environment, and comorbidities.

HIV-1-infected people are approximately 26 times more likely to develop tuberculosis (TB) than HIV-1-uninfected people ( 1 ). This increased risk of developing TB is apparent early after HIV-1 seroconversion: a large study of South African miners found that TB incidence doubled within the first year of HIV-1 infection ( 2 ). Of the 9.6 million reported TB cases in 2014, 1.2 million were coinfected with HIV-1, with 74% of reported HIV-1-infected TB cases being from Africa ( 1 ). The HIV-1 burden in sub-Saharan Africa is particularly high, where 25.8 million people were living with HIV in 2014 and only 41% had access to antiretroviral therapy (ART) ( 3 ). The relatively low ART access may arise in part from a lack of eligibility as determined by local guidelines. It is hoped that more people living with HIV might access ART as a result of the 2015 World Health Organization (WHO) recommendation that ART be initiated for everyone living with HIV at any CD4 cell count ( 4 ). ART reduces TB risk among HIV-1-infected people by 54 to 90% and halves the TB recurrence rate ( 5 ). Despite this risk reduction, HIV-1-infected people established on ART in high TB burden settings remain at higher risk than HIV-1-uninfected people, even in higher CD4 strata ( 6 ).

Since the early 1950s, combination chemotherapy has remained the strongest line of defense against the ancient scourge of tuberculosis (TB). Between the years 2000 and 2015 alone, it was estimated that TB treatment averted 39 million deaths among people without HIV infection and, together with antiretroviral therapy, another 9.6 million deaths among people with HIV infection ( 1 ). Despite these successes, TB continues to exert a terrible toll on humanity. In 2015, TB was estimated to be the cause of 10.4 million new cases and 1.4 million deaths, making Mycobacterium tuberculosis the leading microbial cause of death in the world ( 1 ). The failure to achieve greater control of TB over the past half century is partly attributable to several important limitations of current chemotherapy regimens, including the prolonged treatment durations necessary to prevent relapse after treatment completion and the inability to effectively suppress resistance emergence when treatment is applied on a global scale. These deficiencies are especially notable for current second-line and salvage regimens used to treat drug-resistant TB ( 2 – 4 ), which are also complicated by excessive toxicity, poor tolerability, high cost, and the inconvenience of injections, multiple daily doses, and large pill burdens. As a result, shortening or otherwise simplifying regimens to treat TB without sacrificing efficacy is a major goal of TB drug development research ( 5 ).

The genus Mycobacterium comprises a group of obligately aerobic bacteria that have adapted to inhabit a wide range of intracellular and extracellular environments. Fundamental to this adaptation is the ability to respire and generate energy from variable sources and to sustain metabolism in the absence of growth. The pioneering work of Brodie and colleagues on Mycobacterium phlei established much of the primary information on the electron transport chain and oxidative phosphorylation system in mycobacteria (reviewed in 1 ). Mycobacteria can only generate sufficient energy for growth by coupling the oxidation of electron donors derived from organic carbon catabolism (e.g., NADH, succinate, malate) to the reduction of O2 as a terminal electron acceptor. Mycobacterial genome sequencing revealed that branched pathways exist in mycobacterial species for electron transfer from many low-potential reductants, via quinol, to oxygen ( Fig. 1 ).

Two parallel revolutions were born in the golden era of antibiotics (∼1940 to 1960). One was a revolution in medicine as physicians went to war with microbes. The second was a revolution in biology as microbiologists and geneticists used anti-infectives as tools to reveal how microbes function on a molecular level. Scientists converged on a surprisingly short list of essential biological processes that appeared to make up an Achilles’ heel shared by diverse bacterial pathogens: the biosynthesis of nucleic acids (DNA and RNA), protein, cell walls (peptidoglycan and lipids), and folate. Only later were the far wider dimensions of potential target space appreciated ( 1 ). The discovery of targets led to the development of methods to improve existing antibiotics and find new ones.

Despite the progress made in global tuberculosis (TB) control, TB remains a major global health problem, and drug-resistant TB is a growing threat ( 1 ). Early diagnosis of TB including universal drug susceptibility testing (DST), and systematic screening of contacts and high-risk groups are key components of the End TB Strategy by WHO and partners ( 2 ).

Diagnosis and treatment of latent tuberculosis infection (LTBI) is one of the interventions recommended by the World Health Organization (WHO) to end the TB epidemic worldwide and is one of the elements of the post-2015 End TB Strategy ( 1 ). While several high-income countries, notably the United States and Canada, have implemented and scaled up programs to detect and treat LTBI, developing countries have mostly focused on active TB disease control, a much bigger priority in these settings.

Tuberculosis (TB) remains a global health security risk and a major cause of morbidity and mortality. The TB epidemic continues unabated, with 9.6 million infections occurring globally in 2014, coupled with an overall 1.5 million deaths ( 1 ). Of these infected individuals, 12% were found to have concomitant human immunodeficiency virus (HIV) infection. While prevalence and incidence vary significantly across countries, the annual global incidence has decreased year after year since 2000 by an average of 1.5%. Twenty-two high-burden countries (HBCs) are responsible for 80% of all estimated incident TB cases. Alarmingly, one-third of all TB cases remain undiagnosed (or underreported), and the statistics are significantly worse for drug-resistant TB ( 2 ). Reported multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) cases are inevitably on the rise as increased numbers of individuals are being diagnosed and treated, adherence remains unchecked or unsuccessful, and infection control practices remain suboptimal. Nearly half a million MDR-TB cases are diagnosed annually, representing less than a quarter of estimated incident cases ( 2 ). There is significant work to be done to improve detection of drug-resistant TB and to ensure linkage to appropriate care of patients.

Familial risk of tuberculosis (TB) has been recognized for centuries; indeed, Greek, Arabic, Chinese, and Sanskrit texts are said to include descriptions of the familial nature of disease as early as 600 BCE, and many scholars, including Aristotle (300 BCE), Francastoro (1546), and Marten (1720), explicitly conjectured that this may be because the disease is contagious. However, largely through studies of mono- and dizygotic twin concordance rates, studies of families with Mendelian susceptibility to mycobacterial disease (MSMD), and candidate gene studies performed in the 20th century, it was recognized that susceptibility to TB disease has a substantial host genetic component. Limitations in candidate gene studies and early linkage studies made robust identification of specific loci associated with disease challenging, and few loci have been convincingly associated across multiple populations. Genome-wide association studies (GWAS) and transcriptome-wide association studies, based on microarray (commonly known as genechip) technologies, conducted in the past decade have helped shed some light on pathogenesis, but only a handful of new pathways have been identified. This apparent paradox, of high heritability but few replicable associations, has spurred current large-scale collaborative projects, such as the International Tuberculosis Host Genetics Consortium (ITHGC), that aim to take into account heterogeneity in both host and pathogen genetics, variation in exposure rates, and outcome definitions (referred to as phenotypes by geneticists). Recent studies that also leverage low-cost, high-throughput sequencing to interrogate genetic, transcriptomic, and epigenetic changes in the context of TB are also beginning to be reported.

Tuberculosis has plagued mankind over the centuries and probably accompanied modern Homo sapiens out of Africa. The epidemiological agent of phthisis, also known as “consumption,” reached its epidemic apex during the 18th and 19th centuries. During the industrialization era, the disease was associated with the concentration of labor and poor socioeconomic settings that ultimately favored the spread of this “crowd” pathogen. This high-burden period was then followed by a progressive decline of the death and disease tolls that predated the antibiotic era and the Mycobacterium bovis BCG vaccination. The evolutionary histories of the host and its pathogen are intricately associated, implying that tuberculosis can only be fully understood in the light of H. sapiens origins, migrations, and demography ( 1 ). Excluding these parameters from our analyses might lead us to false conclusions regarding evolution, epidemiology, and pathobiology. In the same line, there is also an urgent need to unravel the genomic features that can explain the contrasted infectivity and transmission observed between Mycobacterium tuberculosis complex (MTBC) lineages ( 2 – 4 ), without neglecting the genetic architecture of the host’s immune system ( 5 ).

The causative agents of human and animal tuberculosis (TB), Mycobacterium tuberculosis and the other members of the M. tuberculosis complex, remain a major cause of human mortality and morbidity and have a massive socioeconomic impact ( 1 ) (http://www.stoptb.org/assets/documents/events/meetings/amsterdam_conference/ahlburg.pdf). According to the latest estimates, around 100 million new tuberculosis (TB) infections, 8.5 million new notified TB cases, and 1.5 million deaths due to TB occur annually ( 2 , 184 ).

The evolution of Mycobacterium tuberculosis toward one of the most dangerous human pathogens is of particular interest for the analysis of the continued importance of tuberculosis as a global disease. While the majority of mycobacterial species are harmless environmental bacteria, M. tuberculosis is able to induce pulmonary lesions and disease in the human host, which represents an essential step for the aerosol transmission of the bacterium to new individuals. The question of when and where M. tuberculosis or one of its progenitors has acquired this faculty has interested scientists for years. It is hypothesized that the ancestors of M. tuberculosis once had an environmental reservoir and gradually evolved to adapt to the life within host cells, leading finally to the feature of getting transmitted from one host to another ( 1 , 2 ). First insights into this issue can be obtained from genomic comparison of M. tuberculosis with related nontuberculous mycobacteria (NTM), also known as atypical mycobacteria or mycobacteria other than tuberculosis (MOTT). Based on 16S rRNA sequence similarity, Mycobacterium marinum and Mycobacterium ulcerans were the currently known closest relatives of M. tuberculosis ( 3 ). In a later study, based on whole-genome sequencing (WGS), Mycobacterium kansasii was found as the closest NTM species of M. tuberculosis ( 4 ), whereas a different WGS study designated M. marinum/M. ulcerans as most closely related to M. tuberculosis, followed by a subgroup containing Mycobacterium haemophilum, Mycobacterium lepromatosis, and Mycobacterium leprae ( 5 ). However, despite the similarities at the DNA and protein level, the genome size differences between the closest NTM species relative to M. tuberculosis are considerable ( 6 ). M. tuberculosis strains harbor a 4.4 MB genome ( 7 – 9 ), whereas the genomes of M. marinum, M. kansasii, and M. ulcerans are larger in size (6.7, 6.4, and 5.8 MB, respectively) ( 10 – 12 ). In contrast, the genomes of M. haemophilum, M. lepromatosis, and M. leprae are smaller in size (4.2, 3.3, and 3.3 MB, respectively) ( 5 , 13 , 14 ), whereby the strong size reduction of the latter two species is due to a common, extensive phase of reductive evolution ( 13 ). It seems conceivable that the individual adaptations of the different mycobacterial species to specific environmental conditions went along with genome downsizing, gene acquisition through horizontal gene transfer, genome rearrangements, and/or recombination.

Mycobacterium tuberculosis possesses a unique cell wall architecture that is distinct from both Gram-negative and Gram-positive bacteria. The cell wall consists of a thick, lipid-rich outer layer composed primarily of mycolic acids ( 1 ) ( Fig. 1 ). This lipid layer lies on top of a layer of peptidoglycan and the polysaccharide arabinogalactan, which, in turn, are anchored to the inner lipid membrane common to all bacteria ( 2 – 4 ). The overall thick waxy coat renders acid-fast (AF) mycobacteria resistant to Gram staining. When stained with alternative dyes, the cell wall is resistant to decolorization with acid alcohol, thus giving these bacteria their sobriquet “acid-fast.” This unique AF property has been the basis for the continuous development of staining procedures over the past century and remains the cornerstone for the diagnosis of tuberculosis (TB), especially in low-income and middle-income countries where more than 90% of TB cases occur ( 5 ). The Ziehl-Neelsen (ZN) stain, also known as the AF stain, which is used in microscopic detection of M. tuberculosis, was originally developed independently by Ziehl and Neelsen, who improved on the early work of Koch, Rindfleisch, and Ehrlich (see below).

The ongoing emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis not only underscores the limitations of our current tuberculosis (TB) control strategies but is also escalating the TB epidemic to a new level. Realizing the imminent threats of MDR- and XDR-TB and the urgency for new TB control measures, the World Health Organization has maintained TB control as high priority and set an ambitious goal of eradicating the disease by 2030 ( 1 ). What remains an urgent need is the development of a shorter-duration combination of antimicrobial drug treatments that is more effective at eradicating drug-susceptible and drug-resistant strains of M. tuberculosis. However, progress toward this goal is hampered by a lack of understanding of factors that contribute to the expression of in vivo drug tolerance by M. tuberculosis, which contributes significantly to the need to treat patients from 6 to 9 months with antimicrobial drug combinations that have toxic side effects. In this review, we discuss the current state of our understanding of the host and pathogen factors that contribute to M. tuberculosis drug tolerance. Moreover, we highlight potential strategies that can be used to improve the efficacy of existing drugs against drug-tolerant M. tuberculosis. These strategies are based on our current knowledge of how and where drug-tolerant bacilli persist and on features of the complex host response that likely limit the penetration of antibiotics. A better understanding of the factors that contribute to the expression of drug tolerance reveals the potential value of adjunctive therapies that can be used to potentiate the effectiveness of existing and future anti-TB drugs.

Mycobacterium tuberculosis is one of the oldest and most successful pathogens in human history due in large part to its coevolution with humans, resulting in an exquisite adaptation by the bacterium to its host. Detection of M. tuberculosis DNA in mummified human remains from both the Old and New World is evidence that tuberculosis (TB) has been part of our history for millennia ( 1 ). In addition, genomic analysis demonstrated that genetic expansion of the mycobacterial repertoire coincided with the geographical expansions of humans, solidifying the evidence that M. tuberculosis has evolved with its host ( 2 ). During this evolution, M. tuberculosis has developed numerous ways to subvert the human immune response ( 3 – 5 ). For instance, a hallmark of M. tuberculosis pathogenicity is its ability to establish a niche in macrophages, the host immune cells that should be the bacterium’s ultimate undoing. Macrophages are a crucial cell subset of the innate immune system whose primary function is to patrol the host and seek out foreign particles. Bacteria and other pathogens are recognized via their pathogen-associated molecular patterns, which initiate a signaling cascade that results in phagocytosis of the pathogen and upregulation of a proinflammatory response. The most notable cytokines produced by macrophages associated with this proinflammatory state in TB are interleukin-1 (IL-1), IL-6, IL-8, IL-12, and tumor necrosis factor (TNF) ( 6 ). Ideally, bacteria are killed and degraded upon phagosome-lysosomal fusion, and as antigen-presenting cells (APCs), macrophages present M. tuberculosis peptides and lipid antigens, ultimately leading to a highly specific adaptive immune response. Therefore, the primary encounter between M. tuberculosis and macrophages dictates the subsequent immune response. In TB, this immune response is focused on containment and eventual eradication of the bacterium in the granuloma.

Protein phosphorylation is known to occur across all three kingdoms of life; however, the study of posttranslational modification in bacteria was neglected for a considerable amount of time. Early attempts to detect its presence were unsuccessful, generating the dogma that protein phosphorylation was a regulatory mechanism that emerged late in evolution to meet the needs of organisms composed of multiple and differentiated cells. The pioneering work of several groups in the 1970s identified protein kinase activity in both Escherichia coli and Salmonella typhimurium ( 1 – 3 ), which soon led to the discovery of the histidine/aspartate kinases of the two-component systems ( 4 , 5 ). The first aspect of this system involves the stimulation of a histidine kinase by a particular environmental or intracellular signal resulting in autophosphorylation on a key histidine residue. The phospho-histidine can then be used as a substrate by the cognate response regulator for its own autophosphorylation on an aspartate residue. The majority of response regulators are DNA binding proteins that trigger expression from target promoters. Unlike the cross-reactivity observed with serine/threonine/tyrosine (Ser/Thr/Tyr) kinases in eukaryotic cell signaling cascades, two-component systems work in isolation, where a given pairing of histidine kinase and response regulator is highly selective for each other via protein-protein interaction.

The transfer of genetic material through successive generations is essential to the survival and evolution of all living organisms, including bacteria. As causative agent of tuberculosis (TB), Mycobacterium tuberculosis must complete successive cycles of transmission, infection, and disease in order to maintain a viable presence in the human population. And, like other pathogens ( 1 ), M. tuberculosis is faced with the extra problem of regulating DNA replication, chromosomal segregation, and cell division while residing in diverse anatomical and cellular loci within its human host—including extra- and intracellular compartments ( 2 , 3 ). Therefore, in addition to the metabolic challenges faced during infection of dynamic and often hostile environments ( 4 , 5 ), M. tuberculosis is likely to encounter multiple stresses that are directly or indirectly genotoxic ( 6 – 8 ). In patients with active TB disease, these stresses might arise from host-derived antimicrobial immune effectors, generation of toxic by-products from host and/or mycobacterial metabolism, changes in intracellular redox potential as a function of shifts in metabolic activity, pH, or oxygen availability, or even exposure to anti-TB drugs. However, given that the number of active TB cases (although devastatingly high in absolute terms) is small relative to the total number of estimated infections ( 9 ), an additional feature of M. tuberculosis is the ability of infecting bacilli to persist for decades in a poorly understood subclinical state ( 10 , 11 ), in some cases reactivating decades later to cause postprimary TB ( 12 , 13 ). Under these conditions, DNA replication and repair pathways are predicted to be essential for preserving the genetic content and viability of bacilli located in lesions characterized by different states of immune activation at various stages throughout the disease cycle ( 14 ).

Approximately 20% of bacterial proteins have functions outside the cytoplasm ( 1 ). Consequently, all bacteria possess protein export pathways that transport proteins made in the cytoplasm beyond the cytoplasmic membrane. These exported proteins may remain in the bacterial cell envelope or be further secreted to the extracellular environment. Many exported proteins function in essential physiological processes. Additionally, in bacterial pathogens, many exported proteins have functions in virulence. Consequently, the pathways that export proteins are commonly essential and/or are important for pathogenesis. Across bacteria, including mycobacteria, there are conserved protein export pathways: the general secretion (Sec) and the twin-arginine translocation (Tat) pathways. Both Sec and Tat pathways are essential to the viability of Mycobacterium tuberculosis and both also contribute to virulence (L. Rank and M. Braunstein, unpublished; 2 – 4 ). In addition to these conserved pathways, bacterial pathogens commonly have specialized protein export systems that are important for pathogenesis due to their role in exporting virulence factors. Mycobacteria also have specialized protein export systems: the SecA2 export pathway and five ESX (type VII) pathways. In this article, we focus on the conserved Sec pathway and the specialized SecA2 pathway, review what is known about their respective exportomes, and discuss their importance during M. tuberculosis replication and persistence within the host.

The identification of ESAT-6 secretion system-1 (ESX-1) as a virulence determinant of Mycobacterium tuberculosis is a major discovery in the history of tuberculosis research. ESX-1 is encoded by a genetic locus known as RD1, which stands for “region of difference” and is one of the deleted regions in the vaccine strain Mycobacterium bovis bacille Calmette-Guérin (BCG) for humans ( 1 ). The first evidence emerges from the finding that the absence of RD1 is responsible for the attenuation of BCG’s virulence ( 2 – 4 ). Introduction of RD1 into BCG is sufficient to induce BCG growth in lung and spleen, granuloma formation in lung, splenomegaly, and inflammation and abscesses in liver and kidney in mice ( 4 ). Conversely, deletion of RD1 in the virulent H37Rv M. tuberculosis strain inactivates the ability of H37Rv to enable rapid bacterial replication in lung and spleen, to cause lung histopathology and death in mice ( 2 , 3 ). Lung sections from infected mice show evidence of macrophage lysis, which is a RD1-dependent process ( 2 ). Consistent with this observation, Lewis et al. describe the requirement of RD1 for H37Rv to grow within and kill human macrophages ( 3 ).

Mycobacterium tuberculosis is the causative agent of human tuberculosis. The bacterium has the capacity to persist in its human host for decades prior to progressing to active disease. In fact, on balance, humans deal with M. tuberculosis infection quite effectively with only an estimated 5 to 10% of those infected actually ending up with clinical disease. However, because of the extraordinary penetrance of this infectious agent across the global population, this accounts for in excess of 1 million deaths due to tuberculosis every year. The combination with HIV in sub-Saharan Africa is catastrophic, and M. tuberculosis is now the leading cause of mortality among individuals living with HIV.

DNA evidence indicates that Mycobacterium tuberculosis and humans have cohabited with one another since the emergence of Homo sapiens as a species ( 1 ). In humans, M. tuberculosis resides chiefly within and amidst cells of the immune system. M. tuberculosis has thus evolved in close physical and functional proximity to host immunity.

The terms “genotype” and “phenotype” were coined by the botanist and geneticist Wilhelm Johannsen at the beginning of the 20th century ( 1 ). Both words have a Greek etymology, meaning “generation of form” and “appearance of form,” respectively. Hierarchically, the genotype predates the phenotype, considering that the genotype is defined as the genetic composition of a living entity, while the phenotype is defined as the perceivable characteristics of a living entity, which result from the interaction between the genetic composition and the environment. From unicellular to multicellular organisms, from bacteria to animals, the key for success, especially to evolve and adapt, lies in diversity. Diversity offers two main advantages: first, variants, exhibiting variety, could have a potential advantage against rapid adverse changes in environment, and, second, variants could potentially interact among themselves (mutualism) and perform more efficiently as a population than as individuals. Therefore, organisms have developed various means of generating and maintaining diversity. Changing the genetic content is a way of generating this diversity even though such changes are less frequent and can potentially be detrimental unless selected. Regardless, genetic diversity has been extensively documented even in monomorphic organisms such as Mycobacterium tuberculosis, and this often has a significant impact on the host-pathogen interaction and stimulation of host immune responses ( 2 – 5 ) as well as treatment outcomes ( 6 ). For example, some pathogens exhibit phase variation whereby diversity is generated by highly mutable loci ( 7 ). Genetic diversity in M. tuberculosis is being reviewed elsewhere ( 287 ) and will not be addressed here. In this review we focus exclusively on nongenetic modes of heterogeneity.

Interactions of bacteria with the human host are, in the vast majority of cases, beneficial for both partners ( 1 ). In fact, humans are dependent on their microbial associates for nutrition, defense, and development ( 1 ). However, a minority of bacteria use the human organism as a vessel to proliferate and spread and, as a consequence, leave behind collateral damage of varying degrees. These so-called pathogens have typically evolved to inhabit niches in the human body with little competition from their commensal counterparts ( 2 ). Many of these human pathogens are intracellular bacteria, meaning that their preferred niche of proliferation and persistence is within human cells. Intracellular pathogens invade phagocytic or nonphagocytic host cells, where they replicate in specialized phagosomal compartments or in the cytosol. After having made their way into their preferred niche, they try to benefit from host nutrients and other metabolites to satisfy their bioenergetic and biosynthetic requirements ( 3 ). The dynamic metabolic interplay between pathogen and host is essential for virulence, disease progression, and infection control.